Human embryonic and induced pluripotent stem cells (PSCs) are defined, in part, by their ability to differentiate into all cell types of the human body. For cardiovascular medicine, PSC-derived cardiomyocytes (PSC-CMs) have the potential to be an unlimited ex vivo source of transplantable cardiac cells for use in regenerative therapies of the heart. PSC-CMs can also be used for cardiac drug discovery and screening, and the development of in vitro models of various genetic heart diseases. These basic and clinical applications of PSC-CMs require the elimination of the undifferentiated cells and non-CMs to yield pure PSC-CM populations. For transplantation purposes, having a well-defined population of cardiomyocytes (CMs) may be critical to optimizing stem cell therapy. Even more critical is the removal of PSCs from PSC-CMs due to their tendency to form teratomas upon transplantation into a mature tissue environment. In vitro, a pure, countable population of CMs is necessary for well-controlled experimentation.
There is, however, currently no widely accepted, non-invasive method for purifying live PSC-CMs. The difficulty with PSC-CM purification lies in the fact that CMs have no established, highly specific surface markers that make their identification and physical separation feasible. They may be identified by immunostaining for intracellular cardiac-specific proteins, but at the cost of rendering the cells nonviable.
To date, a handful of approaches for selecting viable PSC-CMs from stem cells have been demonstrated, but each has its own shortcoming. The ectopic expression of a fluorescence reporter protein under the transcriptional control of a CM-specific promoter, achieved by lentiviral transduction, has been used to identify human embryonic stem cell (hESC) derived cardiomyocytes (hESC-CM) populations with >90% purity. However, the use of transgenic methods in patients raises clinical safety concerns that have not yet been resolved. Labeling with green fluorescent proteins has also been shown to affect actin-myosin interaction and impair the contractile activity of muscle. CMs may be selected based on physical size by means of a Percoll gradient, but purity is limited with this technique.
Yet another method selects cardiac progenitors using a cell surface protein (kinase-insert domain-containing receptor), but the progenitors selected still have the potential to differentiate into smooth muscle cells, cardiac fibroblasts and vascular endothelial cells. A more recent non-genetic approach that identifies live PSC-CMs by using a fluorescent dye to detect the increased mitochondrial content of PSCCMs relative to PSCs and non-CMs has the potential to identify CMs with high specificity. However, the number of CMs isolated by mitochondrial staining was reported to be 60-90% of the number defined by α-actinin staining, suggesting that the method may not be sufficiently sensitive—it may be limited in its ability to identify less mature CM phenotypes. Furthermore, the use of dyes requires additional sample preparation steps that may lead to cell loss and raises potential toxicity issues for cell transplantation.